The goal of this renewal application is to develop a rational basis for employing novel, clinically relevant histone deacetylase inhibitors (HDIs), particularly the benzamide HDI MS-275 as well as SAHA and LAQ824, to enhance the antileukemic potential of fludarabine and other established nucleoside analogs. In the preceding funding period, the mechanism underlying synergistic interactions between the macrocyclic lactone bryostatin 1 or the PKC inhibitor UCN-01 and various antileukemic agents were determined to be elaboration of TNFalpha or inhibition of Chk1 rather than down-regulation/inhibition of protein kinase C; moreover, several clinical trials combining bryostatin 1 or UCN-01 and nucleoside analogs have since been initiated. Subsequently, it has been determined that antileukemic interactions between HDIs and nucleoside analogs such as fludarabine are, if anything, more pronounced than those involving bryostatin 1. Furthermore, such interactions have been related to several recently described pro-apoptotic HDI actions, including a) interruption of the cytoprotective MEK1/2/ERK and Akt modules accompanied by JNK activation; b) enhanced generation of reactive oxygen species (ROS); and c) perturbations in the expression of certain anti-apoptotic (i.e., Mcl-1 and XIAP) and cell cycle (i.e., cyclin D1, p27KIP1)proteins. Significantly, MS-275 treatment markedly increases fludarabine-mediated generation of ceramide, a pro-apoptotic lipid second messenger linked to nucleoside analog-mediated lethality in leukemic cells. The specific aims of this proposal are to 1) define the functional roles of inactivation of survival (e.g., ERK, Akt and stress-related (e.g., JNK) pathways by HDIs in synergistic interactions with fludarabine; 2) clarify the role of enhanced ROS generation by HDIs in these events; 3) characterize the functional role of Mcl-1, XlAP, p27KIP1, and cyclin D1 down-regulation in HDI/fludarabine interactions; 4) determine whether enhanced generation of ceramide and possibly reciprocal changes in levels of sphingosine-1-phosphate play functional roles in antileukemic synergism; 5) extend these findings to primary human leukemia cells and normal hematopoietic progenitors to determine whether a basis for therapeutic selectivity exists. Information derived from these studies may provide a rational foundation for new therapeutic approaches in leukemia in which novel HDIs such as MS-275 are combined with fludarabine or other established nucleoside analogs.